AMP-activated protein kinase (AMPK) is a metabolic enzyme that is essential for

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1 Abstract AMP-activated protein kinase (AMPK) is a metabolic enzyme that is essential for maintaining homeostasis because it contributes to the regulation of energetic processes such as glucose uptake and fatty acid oxidation. Recent studies using Drosophila melanogaster suggest that AMPK signaling may also be linked to neurodegeneration. In this study, we sought to identify genes associated with neurodegeneration that may interact with AMPK metabolic pathways. Drosophila strains with neurodegenerative mutations were screened for interactions with AMPK using the Gal4-UAS system, a method commonly used to examine gene expression. Using this model, strains that result in viable offspring when AMPK is knocked down indicate that the genes corresponding to these strains compensate for the lack of expression of AMPK signaling. We have identified a gene related to neurodegeneration, NMNAT, that partially rescues viability of flies with suppressed AMPK activity. Further research on NMNAT and other genes that show promising results can reveal the potential of AMPK signaling as a new means of therapy for related neurodegenerative and metabolic diseases. 1

2 Introduction AMP-activated protein kinase (AMPK) plays an important role in regulating metabolism and maintaining homeostasis at cellular as well as whole-body levels. The enzyme is expressed ubiquitously throughout an organism s body and is found in tissues of the liver, brain, skeletal muscle, and more. It mainly functions by sensing fluctuations in the energy-transfer compounds ATP and AMP and coordinating numerous catabolic and anabolic processes including glucose uptake and fatty acid metabolism (Mihaylova and Shaw, 2011). As a result, AMPK is an essential gene and is especially important for organisms when experiencing stress such as hypoglycemia or hypoxia (Hardie et al., 2006; Shaw, 2006). Recent studies using Drosophila melanogaster have suggested that AMPK metabolic pathways may also be related to the development of neurodegenerative traits. AMPK levels and localization in neuronal structures have been shown to correlate with the development of diseases such as Alzheimer s disease and Huntington s disease (Salminen et al., 2011; Ju et al., 2014). Additionally, Swick et al. found Figure 1. (B) Wild type neurons expressing Actin. (C) AMPKα mutant showing enlarged plasma membrane domains in neuronal dendrites. (D) Wild-type AMPKα transgene autonomously within neurons completely rescues the dendrite phenotype (from Swick et al,. 2013). that certain fly strains containing mutants for AMPK also experience blebbing, or swelling, in neuronal dendrites; this blebbing is reflective of cell death (Figure 1 from Swick et al., 2013). Although the knockout of AMPK is normally lethal, other studies by Oyenwoke et al. demonstrate that crosses between these knockout flies and other strains with mutations in the TRPML gene result in some rescue of viability. TRPML codes for MCOLN channels which play a role in autophagy and are known to produce neurodegenerative characteristics when abnormal 2

3 (Oyenwoke et al., 2015). As a result, this research suggests that AMPK signaling may be linked to genes that contribute to the development of neurons and affect traits linked to neurodegeneration. Our project sought to build upon these recent findings. The goal of our experiment was to identify whether genes linked to neurodegeneration are also associated with the metabolic pathways of AMPK. We hypothesized that certain lines of Drosophila with neurodegenerative mutations would be capable of rescue when AMPK was knocked down, or when its expression was suppressed. The detection of such genes may help reveal potential targets for the treatment of neurodegenerative diseases through AMPK signaling. Methods The Gal4-UAS system In order to test our hypothesis, we used Drosophila to conduct genetic screens that implemented the Gal-UAS system. This system is commonly applied in biological studies to analyze the expression and interactions of genes. Gal4 is a transcription activator which binds to the promoter sequence UAS. This binding initiates the transcription of the gene following UAS. For our project, we used UAS-AMPK-RNAi for AMPK knockdown. When a fly strain containing UAS-AMPK-RNAi was crossed with Gal4, the UAS would promote transcription of a small hairpin RNA (shrna) that would induce the degradation of the mrna of AMPK by the endogenous RNA-inducing silencing complex (RISC). Since AMPK is an essential gene for viability, Drosophila strains with Gal4 and UAS-AMPK-RNAi would not survive under normal conditions (Duffy, 2002). A depiction of the Gal4-UAS system is shown in Figure 2. 3

4 Figure 2. The Gal4-UAS system (from Rodan, 2015). To test mutations of genes related to neurodegeneration, we crossed Gal4 strains with mutant strains, and we then crossed progeny containing both the Gal4 and the focal neurodegenerative mutation with UAS-AMPK-RNAi strains. If we found progeny from the final cross that had both the mutation and AMPK knockdown, this discovery would indicate that the mutant gene compensated for suppressed AMPK activity. Screening procedure Our genetic screens required that we identify male and virgin female flies, allow them to mate, and score progeny for the applicable visible markers. Virgin females were collected in the mornings and evenings from fly bottles with pupae that were on the verge of enclosing, and these bottles were placed overnight in an incubator set at 18 C to slow the flies growth. Males were obtained from fly vials. Flies were collected by first injecting carbon dioxide gas into the containers to immobilize the flies. They were then placed on a gas pad to ensure that they did not wake up while they were being analyzed under a microscope. Once the necessary males and virgin females were found based on their expected phenotypes, they were placed together in a vial to allow them to mate. Resulting progeny were scored using a similar process as described. 4

5 Additionally, all fly strains involved in the experiment were continuously transferred into new containers in order to maintain the strains. Control and experimental crosses The mutations that we studied were on either the flies second or the third chromosome. In order to make sure that progeny from the first cross had both the mutation and Gal4, we crossed the mutant strains with fly strains that had Gal4 on a different chromosome. Secondchromosome mutants were crossed with a fly strain whose Gal4 expression was driven by a tubulin promoter (Tub-Gal4) because our Tub-Gal4 is on the third chromosome, and thirdchromosome mutants were crossed with a fly strain whose Gal4 expression was driven by an actin promoter (Act-Gal4) because our Act-Gal4 is on the second chromosome. Both of these Gal4 strains are expressed ubiquitously, ensuring that AMPK is knocked down in all tissues by the Gal4-UAS system (Johnson, 2010). We first conducted test crosses in order to ensure that the Gal4-UAS system functioned properly. Positive control crosses were set up using UAS-AMPK-RNAi flies and flies with either Tub-Gal4 or Act-Gal4. Since the Gal4-UAS system would have been implemented, we expected that progeny with Gal4 would not be viable. Additionally, we used negative control crosses between UAS-GFP flies and either Tub-Gal4 or Act-Gal4 flies in order to rule out the possibility of contamination or abnormalities in Gal4. Since RNAi was not present in the negative control crosses, we expected that all progeny, including those with Gal4, would remain viable. Examples of the test crosses using Tub-Gal4 are diagramed in Supplementary Figures 1 and 2. When we conducted the experimental crosses, the mutant fly strains were crossed with either Tub-Gal4 or Act-Gal4 depending on which chromosome the mutation was located on. 5

6 Progeny that had both Gal4 and the mutation were then crossed with UAS-AMPK-RNAi flies. We expected that final progeny that did not have Gal4 would survive and those with both Gal4 and UAS-AMPK-RNAi would not survive. If progeny with Gal4 and UAS-AMPK-RNAi as well as the mutation were identified, our results would imply that the mutation interacted with AMPK pathways; such progeny were noted as rescues. Examples of the experimental crosses using Tub-Gal4 are diagrammed in Supplementary Figure 3. The mutant fly strains that we studied are listed in Table 1. All flies were obtained from the Bloomington Drosophila Stock Center. Strain Identification Number Gene With Mutation LRRK LRRK LRRK PINK PINK PINK PINK PINK PARK PARK PARK tau tau tau NMNAT NMNAT NMNAT 8297 PSN 8299 PSN PSN Par Par Par-1 Table 1. Drosophila strains used in experimental crosses. Identification numbers are from FlyBase (dos Santos et al.). 6

7 Results Tables 2 and 3 list the results from the test crosses and experimental crosses, respectively. The results were obtained from scoring the final progeny of each cross and interpreting their phenotypes. The results from the test crosses met expectations. When Tub-Gal4 flies or Act-Gal4 flies were crossed with UAS-AMPK-RNAi, progeny that had both Gal4 and UAS-AMPK-RNAi were not viable. Additionally, all possible progeny from the crosses with the Gal4 flies and UAS-GFP were viable. Therefore, we established that the Gal4-UAS system was functioning properly. Type of Control Cross Results Positive Tub-Gal4 x UAS-AMPK- RNAi Progeny with both Tub-Gal4 and UAS-AMPK- RNAi were not viable Positive Act-Gal4 x UAS-AMPK- RNAi Progeny with both Act-Gal4 and UAS-AMPK- RNAi were not viable Negative Tub-Gal4 x UAS-GFP All progeny were viable Negative Act-Gal4 x UAS-GFP All progeny were viable Table 2. Results from test crosses 7

8 Strain Identification Number Gene With Mutation Total Number of Progeny Number of Progeny with Rescue Percent Rescue LRRK % LRRK % LRRK % PINK % PINK % PINK % PINK % PINK % PARK % PARK % PARK % tau % tau % tau % NMNAT % NMNAT % NMNAT % 8297 PSN % 8299 PSN % PSN % Par % Par % Par % Table 3. Results from experimental crosses. Strains that resulted in rescue are highlighted. Discussion Of the 23 fly strains that we tested for the experimental crosses, 2 strains (39698 and 24887) showed rescue because they resulted in viable progeny that had both the mutation and AMPK knockdown. Both of these strains had mutations in the NMNAT gene, so our results indicated that the NMNAT gene may interact with AMPK pathways. NMNAT has been previously implicated in Alzheimer s disease and dendritic spine maintenance (dos Santos et al.). 8

9 As a result, our findings suggested a possible linkage between this neurodegenerative gene and the metabolic mechanisms of AMPK. NMNAT now serves as a solid candidate for further characterization in more definitive assays, such as testing for direct protein interactions. It should be noted that although the other fly strains we analyzed did not show rescue, our results do not rule out the possibility that some of these genes may also interact with AMPK. It is possible that the strains we used were not compatible with our Gal4-UAS screening protocol or that we did not find rescue by chance. We will continue our screening process using different fly strains and with repeat testing to thoroughly examine these and other genes related to neurodegeneration. The identification of genes that impact AMPK pathways can have great implications for future studies as it can uncover previously unknown contributors to mechanisms related to neurodegeneration and the regulation of metabolism by AMPK. These discoveries may also drive therapeutic research of AMPK, which may be a potential target for neurodegenerative diseases, and neurodegenerative genes may provide targets for disorders related to AMPK. Although our research is still in its beginning stages, we hope that further study will help to progress our understanding of the relationship between neurodegeneration and AMPK and highlight its contribution to an organism s health. 9

10 References Boissan, M., Dabernat, S., Peuchant, E., Schlattner, U., Lascu, I., Lacombe, M. (2009). The mammalian NM23/NDPK family: from metastasis control to cilia movement. Mol Cell Biochem 329, Duffy, J. (2002). Gal4 System in Drosophila: A Fly Geneticist s Swiss Army Knife. dos Santos, G., Schroeder, A., Goodman, J., Strelets, V., Crosby, M., Thurmond, J., Emmert, D., Gelbart, W. (2015). Gene Dmel\Nmnat. The FlyBase Consortium. Engel, M., Veron, M.., Theisinger, B., Lacombe, M., Seib, T. Dooley, S., Welter, C. (1995). A novel serine/threonine-specific protein phosphotransferase activity of Nm23/nucleosidediphosphate kinase. Eur J Biochem 234, Hardue, D., Hawley, S., Scott, J. (2006). AMP-activated protein kinase development of the energy sensor concept. J physiol 574, Johnson, E., Kazgan, N., Bretz, C., Forsberg L. Hector, C., Worthen, R. Onyenwoke, R., Brenman, J. (2010). Altered metabolism and persistent starvation behaviors caused by reduced AMPK function in Drosophila. PLoS One 5, piiel2799. Ju, T., Chen, H., Chen, Y., Chang, C.P., Chang, C., Chern, Y. (2014). AMPK-α1 functions downstream of oxidative stress to mediate neuronal atrophy in Huntington's disease. Biochim Biophys Acta. 1842(9), Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J., Tanida, I.,Tanaka, K. (2006). Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441,

11 Meley, D., Bauvy, C., Hauben-Weerts, J., Dubbelhuis, P., Helmond, M., Codogno, P., Meijer, A. (2006). AMP-activated protein kinase and the regulation of autophagic proteolysis. Journalism of Biological Chemistry 281, Mihaylova, M. and Shaw, R. (2011). The AMP-activated protein kinase (AMPK) signaling pathway coordinates cell growth, autophagy, & metabolism. Nat Cell Bio 13(9), Onyenwoke, R., Forsberg, L., Liu, L., Williams, T., Alzate O., Brenman, J.(2012). AMPK directly inhibits NDPK through a phosphoserine switch to maintain cellular homeostasis. Molecular Biology of the Cell, 23, Oyenwoke, R., Sexton, J., Yan, F., Díaz, M., Forsberg, L., Major, M., Brenman, J. (2015). The mucolipidosis IV Ca2 channel TRPML1 (MCOLN1) is regulated by the TOR kinase. Biochem J 470(3), Prüßing, K., Voigt, A., Schulz, J. (2013). Drosophila melanogaster as a model organism for Alzheimer s disease. Molecular Neurodegeneration, 8. Rodan, A. (2015). Rodan Lab Research. UT Southwestern Medical Center. Salminen, A., Kaarniranta K., Haapasalo, A., Soininen, H., Hiltunen, M. (2011). AMP-activated protein kinase: a potential player in Alzheimer s disease. J. Neurochem. 118(4), Samari, H., Møller, M., Holden, L., Asmyhr, T., Seglen, P. (2005). Stimulation of hepatocytic AMP-activated protein kinase by okadaic acid and other autophagy-suppressive toxins. Biochemical Journal, 386,

12 Swick, L., Kazgan, N., Onyenwoke, R., Brenman, J. (2013). Isolation of AMP-activated protein kinase (AMPK) alleles required for neuronal maintenance in Drosophila melanogaster. Biology Open 2, Venkatachalam, K., Long, A., Elsaesser, R., Nikolaeya, D., Broadie, K., Montell, C. (2008). Motor deficit in a Drosophila model of Mucolipidosis Type IV due to defective clearance of apoptotic cells, Cell, 135, Williams, T., Brenman, J. (2008). LKB1 and AMPK in cell polarity and division. Trends Cell Biol, 18, Wang, Z., Wilson, W., Fujino, M., Roach, P. (2001). Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Molecular and Cellular Biology, 21,

13 SUPPLEMENTARY INFORMATION Figure 1. Positive control cross with Tub-Gal4 flies ; ; TM3,Sb ( ) x y ; Kr ; CyO ( ) (1 st chromosome) y y y (2 nd chromosome) Kr Kr Kr CyO CyO Cyo (3 rd chromosome) Tub Gal4 TM3, Sb AMPKα RNAi TM3,Sb AMPKα RNAi TM3,Sb (y or ) (Kr or CyO) ; ; These progeny are not expected to survive because the Gal4-UAS system will activate RNAi expression, inhibiting AMPK (y or ) (Kr or CyO) ; ; TM3,Sb These progeny are expected to survive because Gal4 is not present so RNAi will not be expressed. Sb codes for the stubble phenotype, so all progeny should be stubble. 13

14 Figure 2. Negative control cross with Tub-Gal4 flies ; ; TM3,Sb ( ) x y ; UAS GFP UAS GFP ; ( ) (1 st chromosome) y y y (2 nd chromosome) UAS GFP UAS GFP UAS GFP UAS GFP UAS GFP UAS GFP (3 rd chromosome) Tub Gal4 TM3, Sb TM3,Sb TM3,Sb ; UAS GFP (y or ) ; ( or TM3,Sb) All progeny are expected to survive because GFP is nontoxic and is not thought to dramatically affect cell function. 14

15 Figure 3A. Experimental cross between mutant (mut) and Tub-Gal4 flies ; ; TM3,Sb ( ) x y ; mut balancer ; ( ) (1 st chromosome) (2 nd chromosome) mut mut mut y y y balancer balancer balancer (3 rd chromosome) Tub Gal4 TM3, Sb TM3,Sb TM3,Sb Selected progeny: y w ; mut ; Since these progeny do not have Sb, they will have a non-stubble phenotype and will not show ( ) the phenotype of the balancer on the mutation chromosome. Figure 3B. Experimental cross with UAS-AMPKα-RNAi ; Kr ; CyO ( ) x ; mut ; ( ) (1 st chromosome) (2 nd chromosome) Kr CyO mut mut mut Kr CyO y Kr y y CyO (3 rd chromosome) AMPKα RNAi AMPKα RNAi Tub Gal4 TM3,Sb 15

16 (y or ) (Kr or CyO) ; ; ; mut ; (y or ) (Kr or CyO) (y or ) (Kr or CyO) ; ; These progeny are expected to survive because Gal4 is not present so RNAi will not be expressed These progeny are not expected to survive because the Gal4-UAS system will activate RNAi expression, inhibiting AMPK ; mut ; (y or ) (Kr or CyO) These progeny will not survive unless the mutation rescues the flies; if so, further testing will be conducted on the line to determine if the gene acts on AMPK metabolic pathways 16